Substituted 2-aminobenzamide caspase inhibitors and the use thereof

ABSTRACT

The present invention is directed to novel 2-aminobenzamide thereof, represented by the general Formula I:  
                 
 
     where R 1 -R 3 , X, and A-D are defined herein. The present invention also relates to the discovery that compounds having Formula I are potent inhibitors of caspases and apoptotic cell death. Therefore, the inhibitors of this invention can retard or block cell death in a variety of clinical conditions in which the loss of cells, tissues or entire organs occurs.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] This invention is in the field of medicinal chemistry. Inparticular, the invention relates to substituted 2-aminobenzamides andanalogs that are inhibitors of caspases. The invention also relates tothe use of these 2-aminobenzamides and analogs for reducing or treatingapoptotic cell death and/or reducing interleukin 1-β production.

[0003] 2. Description of Background Art

[0004] Organisms eliminate unwanted cells by a process variously knownas regulated cell death, programmed cell death or apoptosis. Such celldeath occurs as a normal aspect of animal development as well as intissue homeostasis and aging (Glucksmann, A., Biol. Rev. CambridgePhilos. Soc. 26:59-86 (1951); Glucksmann, A., Archives de Biologie76:419-437 (1965); Ellis et al., Dev. 112:591-603 (1991); Vaux et al.,Cell 76:777-779 (1994)). Apoptosis regulates cell number, facilitatesmorphogenesis, removes harmful or otherwise abnormal cells andeliminates cells that have already performed their function.Additionally, apoptosis occurs in response to various physiologicalstresses, such as hypoxia or ischemia (PCT published applicationWO96/20721).

[0005] There are a number of morphological changes shared by cellsexperiencing regulated cell death, including plasma and nuclear membraneblebbing, cell shrinkage (condensation of nucleoplasm and cytoplasm),organelle relocalization and compaction, chromatin condensation andproduction of apoptotic bodies (membrane enclosed particles containingintracellular material) (Orrenius, S., J. Internal Medicine 237:529-536(1995)).

[0006] Apoptosis is achieved through an endogenous mechanism of cellularsuicide (Wyllie, A. H., in Cell Death in Biology and Pathology, Bowenand Lockshin, eds., Chapman and Hall (1981), pp. 9-34). A cell activatesits internally encoded suicide program as a result of either internal orexternal signals. The suicide program is executed through the activationof a carefully regulated genetic program (Wylie et al., Int. Rev. Cyt.68: 251 (1980); Ellis et al., Ann. Rev. Cell Bio. 7: 663 (1991)).Apoptotic cells and bodies are usually recognized and cleared byneighboring cells or macrophages before lysis. Because of this clearancemechanism, inflammation is not induced despite the clearance of greatnumbers of cells (Orrenius, S., J. Internal Medicine 237:529-536(1995)).

[0007] Mammalian interleukin-1β (IL-1β) plays an important role invarious pathologic processes, including chronic and acute inflammationand autoimmune diseases (Oppenheim, J. H. et. al. Immunology Today, 7,45-56 (1986)). IL-1β is synthesized as a cell associated precursorpolypeptide (pro-IL-1β) that is unable to bind IL-1 receptors and isbiologically inactive (Mosley et al., J. Biol. Chem. 262:2941-2944(1987)). By inhibiting conversion of precursor IL-1β to mature IL-1β,the activity of interleukin-1 can be inhibited. Interleukin-1βconverting enzyme (ICE) is a protease responsible for the activation ofinterleukin-1β (IL-1β) (Thornberry, N. A., et al., Nature 356: 768(1992); Yuan, J., et al., Cell 75: 641 (1993)). ICE is asubstrate-specific cysteine protease that cleaves the inactiveprointerleukin-1 to produce the mature IL-1. The genes that encode forICE and CPP32 are members of the mammalian ICE/Ced-3 family of geneswhich presently includes at least twelve members: ICE,CPP32/Yama/Apopain, mICE2, ICE4, ICH1, TX/ICH-2, MCH2, MCH3, MCH4,FLICE/MACH/MCH5, ICE-LAP6 and ICE_(re1)III. The proteolytic activity ofthis family of cysteine proteases, whose active site (a cysteineresidue) is essential for ICE-mediated apoptosis, appears critical inmediating cell death (Miura et al., Cell 75: 653-660 (1993)). This genefamily has recently been named caspases (Alnernri, E. S. et. al. Cell,87, 171 (1996), and Thornberry, N. A. et. al. J. Biol. Chem. 272,17907-17911 (1997)) and divided into three groups according to its knownfunctions. Table 1 summarizes these known caspases. TABLE 1 Enzyme*Group I: mediators of inflammation Caspase-1 (ICE) Caspase-4 (ICE_(rel)-II, TX, ICH-2) Caspase-5 (ICE_(rel) -III, TY) Group II: effectors ofapoptosis Caspase-2 (ICH-1, mNEDD2) Caspase-3 (apopain, CPP-32, YAMA)Caspase-7 (Mch-3, ICE-LAP3, CMH-1) Group III: activators of apoptosisCaspase-6 (Mch2) Caspase-8 (MACH, FLICE, Mch5) Caspase-9 (ICE-LAP6,Mch6) Caspase-10

[0008] IL-1 is also a cytokine involved in mediating a wide range ofbiological responses including inflammation, septic shock, woundhealing, hematopoiesis and growth of certain leukemias (Dinarello, C.A., Blood 77:1627-1652 (1991); diGiovine et al., Immunology Today 11:13(1990)).

[0009] Many potent caspase inhibitors have been prepared based on thepeptide substrate structures of caspases. However, in contrast to theirpotency in vitro, no inhibitors with good efficacy (IC₅₀<1 μM) inwhole-cell models of apoptosis have been reported (Thornberry, N. A.Chem. Biol. 5:R97-103 (1998)). Therefore the need exists for cell deathinhibitors that are efficacy in whole-cell models of apoptosis andactive in animal model of apoptosis. These inhibitors thus can beemployed as therapeutic agents to treat disease states in whichregulated cell death and the cytokine activity of IL-1 play a role.

[0010] WO 93/05071 discloses peptide ICE inhibitors with the formula:

Z-Q₂-Asp-Q₁

[0011] wherein Z is an N-terminal protecting group; Q₂ is 0 to 4 aminoacids such that the sequence Q₂-Asp corresponds to at least a portion ofthe sequence Ala-Tyr-Val-His-Asp; Q₁ comprises an electronegativeleaving group.

[0012] WO 96/03982 discloses aspartic acid analogs as ICE inhibitorswith the formula:

[0013] wherein R₂ is H or alkyl; R₃ is a leaving group such as halogen;R₁ is heteroaryl-CO or an amino acid residue.

[0014] U.S. Pat. No. 5,585,357 discloses peptidic ketones as ICEinhibitors with the formula:

[0015] wherein n is 0-2; each AA is independently L-valine or L-alanine;R₁ is selected from the group consisting of N-benzyloxycarbonyl andother groups; R₈, R₉, R₁₀ are each independently hydrogen, lower alkyland other groups.

[0016] Mjalli et al. (Bioorg. Med. Chem. Lett., 3, 2689-2692, 1993)report the preparation of peptide phenylalkyl ketones as reversibleinhibitors of ICE, such as:

[0017] Thornberry et al. (Biochemistry, 33, 3934-3940, 1994) report theirreversible inactivation of ICE by peptide acyloxymethyl ketones:

[0018] wherein Ar is COPh-2,6-(CF₃)₂, COPh-2,6-(CH₃)₂, Ph-F₅ and othergroups.

[0019] Dolle et al. (J. Med. Chem. 37, 563-564, 1994) report thepreparation of P₁ aspartate-based peptideα-((2,6-dichlorobenzoyl)oxy)methyl ketones as potent time-dependentinhibitors of ICE, such as:

[0020] Mjalli et al. (Bioorg. Med. Chem. Lett., 4, 1965-1968, 1994)report the preparation of activated ketones as potent reversibleinhibitors of ICE:

[0021] wherein X is NH(CH₂)₂, OCO(CH₂)₂, S(CH₂)₃ and other groups.

[0022] Dolle et al. (J. Med. Chem. 37, 3863-3866, 1994) report thepreparation of α-((1-phenyl-3-(trifluoromethyl)-pyrazol-5-yl)oxy)methylketones as irreversible inhibitor of ICE, such as:

[0023] Mjalli et al. (Bioorg. Med. Chem. Lett., 5, 1405-1408, 1995)report inhibition of ICE by N-acyl-Aspartic acid ketones:

[0024] wherein XR₂ is NH(CH₂)₂Ph, OCO(CH₂)₂cyclohexyl and other groups.

[0025] Mjalli et al. (Bioorg. Med. Chem. Lett., 5, 1409-1414, 1995)report inhibition of ICE by N-acyl-aspartyl aryloxymethyl ketones, suchas:

[0026] Dolle et al. (J. Med. Chem. 38, 220-222, 1995) report thepreparation of aspartyl α-((diphenylphosphinyl)oxy)methyl ketones asirreversible inhibitors of ICE, such as:

[0027] Graybill et al. (Bioorg. Med. Chem. Lett., 7, 41-46, 1997) reportthe preparation of α-((tetronoyl)oxy)- and α-((tetramoyl)oxy)methylketones as inhibitors of ICE, such as:

[0028] Semple et al. (Bioorg. Med. Chem. Lett., 8, 959-964, 1998) reportthe preparation of peptidomimetic aminomethylene ketones as inhibitorsof ICE, such as:

SUMMARY OF THE INVENTION

[0029] The invention relates to compound of Formulae I, II and III:

[0030] wherein

[0031] R₁ is an optionally substituted alkyl or hydrogen;

[0032] R₃ is an N-protecting group;

[0033] R₂ is hydrogen or optionally substituted alkyl;

[0034] A is CR₆ or nitrogen;

[0035] B is CR₇ or nitrogen;

[0036] C is CR₈ or nitrogen;

[0037] D is CR₉ or nitrogen;

[0038] provided that not more than two of A, B, C or D is nitrogen; andR₆-R₉ independently are hydrogen, halo, C₁-C₆ haloalkyl, C₆-C₁₀ aryl,C₄-C₇ cycloalkyl, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₆-C₁₀aryl(C₁-C₆)alkyl, C₆-C₁₀ aryl(C₂-C₆)alkenyl, C₆-C₁₀ aryl(C₂-C₆)alkynyl,C₁-C₆ hydroxyalkyl, nitro, amino, cyano, C₁-C₆ acylamino, hydroxy, C₁-C₆acyloxy, C₁-C₆ alkoxy, alkylthio, or carboxy; or

[0039] one of R₆ and R₇, or R₇ and R₈, or R₈ and R₉ are taken togetherwith the carbon atoms to which they are attached to form a carbocycle orheterocycle;

[0040] E is CR₁₄, nitrogen, oxygen or sulfur;

[0041] F is CR₁₅, nitrogen, oxygen or sulfur;

[0042] G is C₁₆, nitrogen, oxygen or sulfur;

[0043] provided that only one of E, F, G is nitrogen, oxygen or sulfur;where R₁₄-R₁₆ are independently hydrogen, halo, C₁-C₆ haloalkyl, C₆-C₁₀aryl, C₄-C₇ cycloalkyl, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,C₆-C₁₀ aryl(C₁-C₆)alkyl, C₆-C₁₀ aryl(C₂-C₆)alkenyl, C₆-C₁₀aryl(C₂-C₆)alkynyl, C₁-C₆ hydroxyalkyl, nitro, amino, cyano, C₁-C₆acylamino, hydroxy, C₁-C₆ acyloxy, C₁-C₆ alkoxy, alkylthio, or carboxy;or

[0044] one of R₁₄ and R₁₅, or R₁₅ and R₁₆, are taken together with thecarbon atoms to which they are attached to form a carbocycle orheterocycle;

[0045] Q represents an optionally substituted saturated or partiallysaturated carbocycle or heterocycle;

[0046] X is a peptide of 1-4 amino acids or a bond; and

[0047] Y is a peptide of 1-4 amino acids or a bond.

[0048] The invention relates to the discovery that the compoundsrepresented by Formulae I, II and III are inhibitors of caspases. Theinvention also relates to the use of the compounds of the invention forreducing, preventing or treating maladies in which apoptotic cell deathis either a causative factor or a result. Examples of uses for thepresent invention include protecting the nervous system following focalischemia and global ischemia; treating neurodegenerative disorders suchas Alzheimer's disease, Huntington's Disease, prion diseases,Parkinson's Disease, multiple sclerosis, amyotrophic lateral sclerosis,ataxia, telangiectasia, and spinobulbar atrophy; treating heart diseaseincluding myocardial infarction, congestive heart failure andcardiomyopathy; treating retinal disorders; treating autoimmunedisorders including lupus erythematosus, rheumatoid arthritis, type Idiabetes, Sjögren's syndrome and glomerulonephritis; treating polycystickidney disease and anemia/erythropoiesis; treating immune systemdisorders, including AIDS and SCIDS; treating or ameliorating sepsis ormulti-organ failure in an animal; reducing or preventing cell, tissueand organ damage during transplantation; reducing or preventing cellline death in industrial biotechnology; reducing or preventing alopecia(hair loss); reducing the premature death of skin cells; treating orameliorating apoptotic cell death in acute pancreatitus; treating orpreventing the inflammatory response in psoriasis or inflammatory boweldisease; and treating or ameliorating organ apoptosis after burn injury.

[0049] The present invention provides pharmaceutical compositionscomprising a compound of Formulae I, II and III in an effective amountto reduce apoptotic cell death in an animal.

[0050] The present invention also provides preservation or storagesolutions for mammalian organs or tissue, or growth media for mammalianor yeast cells, wherein an effective amount of a compound of Formula I,II and III is included in said solutions or media in order to reduceapoptotic cell death in said organs, tissue or cells.

[0051] The invention also relates to the use of caspase inhibitors fortreating, ameliorating, and preventing non-cancer cell death duringchemotherapy and radiation therapy and for treating and ameliorating theside effects of chemotherapy and radiation therapy of cancer.

[0052] In particular, the invention relates to a method of treating,ameliorating or preventing oral mucositis, gastrointestinal mucositis,bladder mucositis, proctitis, bone marrow cell death, skin cell deathand hair loss resulting from chemotherapy or radiation therapy of cancerin an animal, comprising administering to the animal in need thereof aneffective amount of a caspase inhibitor.

DETAILED DESCRIPTION OF THE INVENTION

[0053] The inhibitors of caspases and apoptotic cell death of thepresent invention are compounds having the general Formulae I, II andIII:

[0054] or pharmaceutically acceptable salts or prodrugs thereof,wherein:

[0055] R₁ is an optionally substituted alkyl or hydrogen;

[0056] R₃ is a N-protecting group including t-butyloxycarbonyl, acetyl,and benzyloxycarbonyl;

[0057] R₂ is hydrogen or optionally substituted alkyl;

[0058] A is CR₆ or nitrogen;

[0059] B is CR₇ or nitrogen;

[0060] C is CR₈ or nitrogen;

[0061] D is CR₉ or nitrogen; provided that not more than two of A, B, Cor D is nitrogen; and R₆-R₉ independently are hydrogen, halo, C₁-C₆haloalkyl, C₆-C₁₀ aryl, C₄-C₇ cycloalkyl, C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₆-C₁₀ aryl(C₁-C₆)alkyl, C₆-C₁₀ aryl(C₂-C₆)alkenyl,C₆-C₁₀ aryl(C₂-C₆)alkynyl, C₁-C₆ hydroxyalkyl, nitro, amino, cyano,C₁-C₆ acylamino, hydroxy, C₁-C₆ acyloxy, C₁-C₆ alkoxy, alkylthio, orcarboxy; or

[0062] one of R₆ and R₇, or R₇ and R₈, or R₈ and R₉ are taken togetherwith the carbon atoms to which they are attached to form a carbocycle orheterocycle;

[0063] E is C₁₄, nitrogen, oxygen or sulfur;

[0064] F is C₁₅, nitrogen, oxygen or sulfur;

[0065] G is C₁₆, nitrogen, oxygen or sulfur; provided that only one ofE, F, G is nitrogen, oxygen or sulfur; where R₁₄-R₁₆ are independentlyhydrogen, halo, C₁-C₆ haloalkyl, C₆-C₁₀ aryl, C₄-C₇ cycloalkyl, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₆-C₁₀ aryl(C₁-C₆)alkyl, C₆-C₁₀aryl(C₂-C₆)alkenyl, C₆-C₁₀ aryl(C₂-C₆)alkynyl, C₁-C₆ hydroxyalkyl,nitro, amino, cyano, C₁-C₆ acylamino, hydroxy, C₁-C₆ acyloxy, C₁-C₆alkoxy, alkylthio, or carboxy; or

[0066] one of R₁₄ and R₁₅, or R₁₅ and R₁₆ are taken together with thecarbon atoms to which they are attached to form a carbocycle orheterocycle;

[0067] Q represents an optionally substituted saturated or partiallysaturated carbocycle or heterocycle;

[0068] X is a peptide of 1-4 amino acids or a bond; And

[0069] Y is a peptide of 1-4 amino acids or a bond. Where X or Y is oneamino acid, it may be any one of the common 20 amino acids e.g., Ala,Val, Leu, Ile, Pro, Phe, Trp, Met, Gly, Ser, Thr, Cys, Tyr, Asp, Asn,Glu, Asn, Lys, Arg and His. Where X is a peptide, it may be Asp-Glu,Asp-Ala, Asp-Phe, Val-Glu, Leu-Glu, Thr-Glu, Ile-Glu, Tyr-Glu, Trp-Glu.Where Y is a peptide, it may be Glu-His, Glu-Ile, Glu-Thr, Glu-Val,Glu-Phe, Thr-His, Val-His, Ala-His and Glu-Pro.

[0070] With respect to R₁, preferred alkyl groups are C₁₋₆ alkyl groups,e.g. methyl, ethyl, propyl, isopropyl, isobutyl, pentyl and hexylgroups; and substituted C₁-₆ alkyl groups, e.g. CH₂OCH₃ and CH₂OCOCH₃(AM).

[0071] The invention relates to the discovery that the compoundsrepresented by Formulae I, II and III are inhibitors of caspases. Theseinhibitors slow or block cell death in a variety of clinical conditionsand industrial applications in which the loss of cells, tissues orentire organs occurs. Therefore, the invention is also related tomethods of treating, preventing or reducing conditions in whichapoptosis plays a role. These conditions are more fully described below.

[0072] The methods comprise administering to an animal in need of suchtreatment an inhibitor of the present invention, or a pharmaceuticallyacceptable salt or prodrug thereof, in an amount effective to inhibitapoptotic cell death.

[0073] Preferred embodiments of the compounds of Formulae I, II and IIIthat may be employed as inhibitors of caspases are represented byFormula IV:

[0074] or pharmaceutically acceptable salts or prodrugs thereof whereinR₁ R₂-, R₆-R₉ and X are as defined previously with respect to Formula I.

[0075] Examples of bridges formed by R₆ and R₇, or R₇ and R₈, or R₈ andR₉ taken together are —OCH₂O—, —OCF₂O—, —(CH₂)₃—, —(CH₂)₄—, —OCH₂CH₂O—,—CH₂N(R₁₃)CH₂—, —CH₂CH₂N(R₁₃)CH₂—, —CH₂N(R₁₃)CH₂CH₂— and —CH═CH—CH═CH—;where R₁₃ is hydrogen, alkyl or cycloalkyl.

[0076] R₁₀ is hydrogen, C₁-C₆ alkoxy, C₁-C₆ alkyl, C₁-C₆ haloalkyl,C₆-C₁₀ aryl, C₄-C₇ cycloalkyl, C₆-C₁₀ aryl(C₁-C₆)alkyl, benzyloxy,substituted benzyloxy, or NR₁₁R₁₂, wherein R₁₁ and R₁₂ independently arehydrogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₆-C₁₀ aryl, C₄-C₇ cycloalkyl,C₆-C₁₀ aryl(C₁-C₆)alkyl, or R₁₁ and R₁₂ are combined to form aheterocyclic ring system including pyrrolidine, piperidine, piperazine,or morpholine.

[0077] Preferred R₁ is H, Me, Et, t-Bu or AM. Preferred R₂ isfluoromethyl, acyloxymethyl, arylacyloxymethyl and aminomethyl.Preferred R₁₀ is benzyloxy and substituted benzyloxy. Preferred X is apeptide of 1-2 amino acids or a bond.

[0078] Exemplary preferred inhibitors of caspases having Formulae I-IVinclude, without limitation:

[0079] 2-(Z-amino)benzoyl-Asp-fmk,

[0080] 2-(Z-amino)-3-methylbenzoyl-Asp-fmk,

[0081] 2-(Z-amino)-3,5-dimethylbenzoyl-Asp-fmk,

[0082] 2-(Z-amino)-4-chlorobenzoyl-Asp-fmk,

[0083] 2-(Z-amino)-5-chlorobenzoyl-Asp-fmk,

[0084] 2-(Z-amino)-5-fluorobenzoyl-Asp-fmk,

[0085] 2-(Z-amino)-6-fluorobenzoyl-Asp-fmk,

[0086] cis-2-(Z-amino)-cyclohexanecarboxyl-Asp-fmk,

[0087] 2-(Z-amino)-5-methylbenzoyl-Asp-fmk,

[0088] 2-(Z-amino)-6-methylbenzoyl-Asp-fmk,

[0089] 2-(Z-amino)-6-chlorobenzoyl-Asp-fmk,

[0090] 2-(Z-amino)-3-methoxybenzoyl-Asp-fmk,

[0091] 3-(Z-amino)thiophene-2-carboxyl-Asp-fmk,

[0092] 3-(methoxycarbonylamino)thiophene-2-carboxyl-Asp-fmk,

[0093] cis-2-(Z-amino)cyclopentanecarboxyl-Asp-fmk,

[0094] trans-2-(Z-amino)cyclopentanecarboxyl-Asp-fmk,

[0095] 2-(Z-amino)benzoyl-Asp-DCB-methylketone,

[0096] methoxycarbonyl-Val-(2-aminobenzoyl)-Asp-fmk,

[0097] Z-Glu-(2-aminobenzoyl)-Asp-fmk, and

[0098] Z-Val-(2-aminobenzoyl)-Asp-fmk.

[0099] where Z is benzyloxycarbonyl, fmk is fluoromethylketone and DCBis 2,6-dichlorobenzoyloxy.

[0100] Useful aryl groups are C₆₋₁₄ aryl, especially C₆₋₁₀ aryl. TypicalC₆₋₁₄ aryl groups include phenyl, naphthyl, phenanthrenyl, anthracenyl,indenyl, azulenyl, biphenyl, biphenylenyl and fluorenyl groups.

[0101] Useful cycloalkyl groups are C₃₋₈ cycloalkyl. Typical cycloalkylgroups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl andcycloheptyl.

[0102] Useful saturated or partially saturated carbocyclic groups arecycloalkyl groups as defined above, as well as cycloalkenyl groups, suchas cyclopentenyl, cycloheptenyl and cyclooctenyl.

[0103] Useful halo or halogen groups include fluorine, chlorine, bromineand iodine.

[0104] Useful alkyl groups include straight-chained and branched C₁₋₁₀alkyl groups, more preferably C₁₋₆ alkyl groups. Typical C₁₋₁₀ alkylgroups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl,tert-butyl, 3-pentyl, hexyl and octyl groups. Also contemplated is atrimethylene group substituted on two adjoining positions on the benzenering of the compounds of the invention.

[0105] Useful arylalkyl groups include any of the above-mentioned C₁₋₁₀alkyl groups substituted by any of the above-mentioned C₆₋₁₄ arylgroups. Useful values include benzyl, phenethyl and naphthylmethyl.

[0106] Useful haloalkyl groups include C₁₋₁₀ alkyl groups substituted byone or more fluorine, chlorine, bromine or iodine atoms, e.g.fluoromethyl, difluoromethyl, trifluoromethyl, pentafluoroethyl,1,1-difluoroethyl, chloromethyl, chlorofluoromethyl and trichloromethylgroups.

[0107] Useful alkoxy groups include oxygen substituted by one of theC₁₋₁₀ alkyl groups mentioned above.

[0108] Useful alkylthio groups include sulphur substituted by one of theC₁₋₁₀ alkyl groups mentioned above. Also included are the sulfoxides andsulfones of such alkylthio groups.

[0109] Useful acylamino groups are any C₁₋₆ acyl (alkanoyl) attached toan amino nitrogen, e.g. acetamido, propionamido, butanoylamido,pentanoylamido, hexanoylamido as well as aryl-substituted C₂₋₆substituted acyl groups.

[0110] Useful acyloxy groups are any C₁₋₆ acyl (alkanoyl) attached to anoxy (—O—) group, e.g. formyloxy, acetoxy, propionoyloxy, butanoyloxy,pentanoyloxy, hexanoyloxy and the like.

[0111] Useful arylacyloxy groups include any of the aryl groupsmentioned above substituted on any of the acyloxy groups mentionedabove, e.g. 2,6-dichlorobenzoyloxy, 2,6-difluorobenzoyloxy and2,6-di-(trifluoromethyl)-benzoyloxy groups.

[0112] Useful amino groups include —NH₂, —NHR₁₁, and —NR₁₁R₁₂, whereinR₁₁ and R₁₂ are C₁₋₁₀ alkyl or cycloalkyl groups as defined above.

[0113] Useful saturated or partially saturated heterocyclic groupsinclude tetrahydrofuranyl, pyranyl, piperidinyl, piperizinyl,pyrrolidinyl, imidazolidinyl, imidazolinyl, indolinyl, isoindolinyl,quinuclidinyl, morpholinyl, isochromanyl, chromanyl, pyrazolidinylpyrazolinyl, tetronoyl and tetramoyl groups.

[0114] Useful heteroaryl groups include any one of the following:thienyl, benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl,pyranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxanthiinyl,2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl,pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl,indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, phthalzinyl,naphthyridinyl, quinozalinyl, cinnolinyl, pteridinyl, carbazolyl,β-carbolinyl, phenanthridinyl, acrindinyl, perimidinyl, phenanthrolinyl,phenazinyl, isothiazolyl, phenothiazinyl, isoxazolyl, furazanyl,phenoxazinyl, 1,4-dihydroquinoxaline-2,3-dione, 7-aminoisocoumarin,pyrido[1,2-a]pyrimidin-4-one, 1,2-benzoisoxazol-3-yl, benzimidazolyl,2-oxindolyl and 2-oxobenzimidazolyl. Where the heteroaryl group containsa nitrogen atom in a ring, such nitrogen atom may be in the form of anN-oxide, e.g. a pyridyl N-oxide, pyrazinyl N-oxide, pyrimidinyl N-oxideand the like.

[0115] Optional substituents include one or more alkyl; halo; haloalkyl;cycloalkyl; aryl optionally substituted with one or more lower alkyl,halo, haloalkyl or heteroaryl groups; aryloxy optionally substitutedwith one or more lower alkyl, halo, haloalkyl or heteroaryl groups;aralkyl; heteroaryl optionally substitued with one or more lower alkyl,haloalkyl and aryl groups; heteroaryloxy optionally substitued with oneor more lower alkyl, haloalkyl and aryl groups; alkoxy; alkylthio;arylthio; amino; acyloxy; arylacyloxy optionally substitued with one ormore lower alkyl, halo alkyl and aryl groups; Diphenylphosphinyloxyoptionally substituted with one or more lower alkyl, halo or haloalkylgroups; heterocyclo optionally substitued with one or more lower alkyl,halo alkyl and aryl groups; heterocycloalkyloxy optionally substituedwith one or more lower alkyl, halo alkyl and aryl groups; partiallyunsaturated heterocycloalkyl optionally substitued with one or morelower alkyl, halo alkyl and aryl groups; or partially unsaturatedheterocycloalkyloxy optionally substitued with one or more lower alkyl,halo alkyl and aryl groups. Particular examples of such optionalsubstituents that may be present at R₂ include, without limitation,3-pyrazolyloxy optionally substituted at the 2, 4 and 5-positions withlower alkyl; 3-(1-phenyl-3-trifluoromethyl)pyrazolyloxy;2,6-di(trifluoromethyl)benzoyloxy; 2,6-dimethylbenzoyloxy,pentafluorophenoxy; 2,6-dichlorobenzoyloxy;2-(3-(2-imidazolyl)naphthyl)oxy; diphenylphosphinyloxy; tetronyloxy; andtetramoyloxy.

[0116] Certain of the compounds of the present invention may exist asstereoisomers including optical isomers. The invention includes allstereoisomers and both the racemic mixtures of such stereoisomers aswell as the individual enantiomers that may be separated according tomethods that are well known to those of ordinary skill in the art.

[0117] Examples of pharmaceutically acceptable addition salts includeinorganic and organic acid addition salts such as hydrochloride,hydrobromide, phosphate, sulphate, citrate, lactate, tartrate, maleate,fumarate, mandelate and oxalate; and inorganic and organic base additionsalts with bases such as sodium hydroxy andTris(hydroxymethyl)aminomethane (TRIS, tromethane).

[0118] Examples of prodrugs include compounds of Formulae I-IV whereinR₁ is an alkyl group or substituted alkyl group such as CH₂OCH₃ andCH₂OCOCH₃ (AM ester).

[0119] The invention is also directed to a method for treating disordersresponsive to the inhibition of caspases in animals suffering thereof.Particular preferred embodiments of compounds for use in the method ofthis invention are represented by previously defined Formulae I-IV.

[0120] The compounds of this invention may be prepared using methodsknown to those skilled in the art. Specifically, compounds with FormulaeI-IV can be prepared as illustrated by exemplary reactions in Scheme 1.The intermediate 1 was prepared according to Revesz et al. (TetrahedronLett. 35, 9693-9696, 1994). Coupling of 1 with a N-protected2-aminobenzoic acid, which is either commercially available or which canbe prepared from a commercially available 2-aminobenzoic acid, such as2-Z-aminobenzoic acid, gave amide 2. Oxidation of 2 by Dess-Martinreagent according to Revesz et al. (Tetrahedron Lett. 35, 9693-9696,1994) gave 3. Acid catalyzed cleavage of the ester gave the free acid 4.

[0121] An important aspect of the present invention is the discoverythat compounds having Formulae I-IV are inhibitors of caspases.Therefore, these inhibitors are expected to slow or block cell death ina variety of clinical conditions in which the loss of cells, tissues orentire organs occurs.

[0122] The cell death inhibitors of the present invention can be used toreduce or prevent cell death in the nervous system (brain, spinal cord,and peripheral nervous system) under various conditions of ischemia andexcitotoxicity, including, but not limited to, focal ischemia due tostroke and global ischemia due to cardiac arrest, as well as spinal cordinjury (Emery et al. J. Neurosurgery, 89: 911-920 (1998)). Oneparticular usage is to treat the effects of oxygen deprivation which canoccur during the birth of infants in high-risk labors or drowning. Thecell death inhibitors can also be used to reduce or prevent cell deathin the nervous system due to traumatic injury (such as head trauma),viral infection or radiation-induced nerve cell death (for example, as aside-effect of cancer radiotherapy). The cell death inhibitors can alsobe used to reduce or prevent cell death in a range of neurodegenerativedisorders, including but not limited to Alzheimer's disease (Mattson etal. Brain Res. 807: 167-176 (1998)), Huntington's Disease, Parkinson'sDisease, multiple sclerosis, amyotrophic lateral sclerosis, andspinobulbar atrophy. The in vivo neuroprotective properties of celldeath inhibitors of the invention can be tested in a rat transient focalbrain ischemia model (Xue et al., Stroke 21: 166 (1990)). The cell deathinhibitors may also be used to treat or ameliorate cell death in acutebacterial meningitis.

[0123] The cell death inhibitors of the invention can be used to reduceor prevent cell death in any condition which potentially results in thedeath of cardiac muscle (Black et al., J. Mol. Cel. Card. 30: 733-742(1998) and Maulik et al. Free Radic. Biol. Med. 24: 869-875 (1998)).This includes myocardial infarction due to myocardial ischemia andreperfusion, congestive heart failure and cardiomyopathy. One particularapplication is to reduce or prevent myocardial cell death as occurs incertain viral infections of the heart.

[0124] The in vivo activity of the cell death inhibitors of theinvention can be tested using the “mouse liver apoptosis” modeldescribed by Rodriguez et al. (Rodriguez et al., J. Exp. Med.,184:2067-2072 (1996)). In this model, mice are treated intravenously(IV) with an antiFas antibody which induces massive apoptosis in theliver and other organs, leading to generalized organ failure and death.This model is useful for indirectly testing the systemic bioavailabilityof the cell death inhibitors of the invention, as well as their in vivoanti-apoptotic properties. The cell death inhibitors of the inventiontherefore can be used to reduce or prevent apoptosis of liver cells(Jones et al. Hepatology 27: 1632-42 (1998)) such as in sepsis (Jaeschkeet al. J. Immunol. 160: 3480-3486 (1998)) and hereditary tyrosinemiatype 1 (HT1) (Kubo et al. Prov. Natl. Acad. Sci. USA, 95: 9552-9557(1998). The cell death inhibitors of the invention also can be used totreat hepatitis (Suzuki, Proc. Soc. Exp. Biol. Med. 217: 450-454(1998)); treat or ameliorate apoptotic cell death in acute pancreatitus;and treat or ameliorate organ apoptosis after burn injury.

[0125] The cell death inhibitors of the invention can be used to reduceor prevent cell death of retinal neurons (Kermer et al. J. Neurosci. 18:4656-4662 (1998) and Miller et al. Am. J. Vet. Res. 59: 149-152 (1998))as can occur in disorders which increase intraocular pressure (such asglaucoma) or retinal disorders associated with the aging process (suchas age-related macular degeneration). The inhibitors can also be used totreat hereditary degenerative disorders of the retina, such as retinitispigmentosa.

[0126] The cell death inhibitors of the invention can also be used toreduce or prevent cell death in the kidney. This includes renalamyloidosis (Hiraoka et al. Nippon Jinzo Gakkai Shi, 40: 276-83 (1998)),acute renal failure (Lieberthal et al. Semin Nephrol. 18: 505-518(1998)), murine tubular epithelial cell death induced by cyclosporine A(Ortiz et al. Kidney International Supp. 68: S25-S29 (1998)) andHIV-induced nephropathy (Conaldi et al. J. Clin. Invest. 102: 2041-2049(1998)).

[0127] The cell death inhibitors of the invention can also be used toreduce or prevent cell death of buccal mucosa due to chronic alcoholingestion (Slomiany et al. Biochem. Mol. Biol. Int. 45: 1199-1209(1998)).

[0128] The cell death inhibitors of the invention can also be used toreduce or prevent cell death in plants (Richberg et al. Curr. Opin.Plant Biol. 1: 480-485 (1998)), such as plant cell death due topathogens (Pozo et al. Curr. Biol. 8: 1129-1132 (1998) and Greenberg etal. Cell, 77: 551-563 (1994)).

[0129] The cell death inhibitors of the invention can also be used toreduce or prevent cell death due to radiation andultraviolet-irradiation (Sheikh et al. Oncogene, 17: 2555-2563 (1998)).

[0130] The cell death inhibitors of the invention can also be used toreduce or prevent apoptotic death of bone marrow cells inmyelodysplastic syndromes (MDS) (Mundle et al., Am. J. Hematol. 60:36-47 (1999)).

[0131] The cell death inhibitors of the invention can also be used toreduce or prevent premature death of cells of the immune system, and areparticularly useful in treating immune deficiency disorders, such asacquired immune deficiency syndrome (AIDS), severe combined immunedeficiency syndrome (SCIDS) and related diseases. The cell deathinhibitors can also be used to treat radiation-induced immunesuppression.

[0132] Transplantation of human organs and tissues is a common treatmentfor organ failure. However, during the transplantation process, thedonor organ or tissue is at risk for cell death since it is deprived ofits normal blood supply prior to being implanted in the host. Thisischemic state can be treated with cell death inhibitors by infusioninto the donor organ or tissue, or by direct addition of the cell deathinhibitors to the organ/tissue storage medium. Cell death inhibitors mayalso be used to reduce or prevent cell death in the donor organ/tissueafter it has been transplanted to protect it from the effects ofreperfusion injury and/or effects of host immune cells which kill theirtargets by triggering apoptosis. The cytoprotective effects of celldeath inhibitors can also be used to prevent the death of human oranimal sperm and eggs used in in vitro fertilization procedures. Theseinhibitors can be used during the harvesting process and can also beincluded in the storage medium.

[0133] Mammalian cell lines, insect cells and yeast cells are commonlyused to produce large amounts of recombinant proteins (such asantibodies, enzymes or hormones) for industrial or medicinal use. Thelifespan of some of these cell lines is limited due to growthconditions, the nature of the recombinant molecule being expressed (someare toxic) and other unknown factors. The lifespans of industrial celllines can be extended by including these cell death inhibitors in thegrowth medium in a concentration range of 1-100 μM.

[0134] The factors governing hair growth and loss are largely unknown.There is some evidence, however, that hair follicle regression (referredto as catagen) may be due at least partially to apoptosis. Therefore, itis contemplated that the cell death inhibitors of the present inventioncan be used to treat hair loss that occurs due to various conditions,including but not limited to male-pattern baldness, radiation-induced orchemotherapy-induced hair loss, and hair loss due to emotional stress.There is also evidence that apoptosis may play a role in the loss ofhair color. Therefore, it is contemplated that the cell death inhibitorsof the present invention can also be used in treating or preventingcases of premature graying of the hair.

[0135] The death of skin epithelial cells can occur after exposure tohigh levels of radiation, heat or chemicals. It is contemplated that thecell death inhibitors of the present invention can be used to treat,reduce or prevent this type of skin damage. In one particularapplication, the cell death inhibitors can be applied as part of atopical formulation, e.g. an ointment, to treat acute over-exposure tothe sun and to prevent blistering and peeling of the skin.

[0136] Goldberg et al. (Nature Genetics 13: 442-449 (1996)) reportedrecently that huntingtin, a protein product of Huntington's disease (HD)gene, can be cleaved by CPP32 but not ICE. The mutation underlying HD isan expansion of a CAG trinucleotide at the 5′ end of the HD gene. Thetrinucleotide expansion exceeding 36 repeats is associated with theclinical presentation of HD. The CAG expansion promotes cleavage ofhuntingtin by CPP32, thus links the role of CPP32 in the apoptotic celldeath in HD. Compounds of the present invention with CPP32 inhibitingactivity will be useful in blocking CPP32 induced apoptotic cell death,thus in preventing and treating HD and other disorders characterized byexpansion of trinucleotide repeats such as myotonic dystrophy, fragile Xmental retardation, spinobulbar muscular atrophy, spinocerebellar atoxiatype I and Dentato-Rubro pallidoluysian atrophy.

[0137] The invention relates to a method of treating, ameliorating orpreventing oral mucositis, gastrointestinal mucositis, bladdermucositis, proctitis, bone marrow cell death, skin cell death and hairloss resulting from chemotherapy or radiation therapy of cancer in ananimal, comprising administering to the animal in need thereof aneffective amount of a caspase inhibitor.

[0138] When animals are treated with chemotherapeutic agents and/orradiation to kill cancer cells, an unwanted side effect is the apoptoticdeath of rapidly dividing non-cancer cells. Such non-cancer cellsinclude cells of the gastrointestinal tract, skin, hair, and bone marrowcells. According to the present invention, caspase inhibitors areadministered to such non-cancer cells to prevent apoptosis of suchcells. In a preferred embodiment, the caspase inhibitors areadministered locally, e.g. to the gastrointestinal tract, mouth, skin orscalp to prevent apoptosis of the gastrointestinal, mouth, skin or haircells but allowing for the death of the cancer cells. Thus, in oneexample, it is possible to treat brain cancer with chemotherapy orradiation therapy and protect the outer skin, hair cells,gastrointestinal tract and bone marrow by local administration of acaspase inhibitor. In the case of oral mucositis, the caspase inhibitorcan be applied, for example, in the form of a mouth wash or mouth rinse,in a gel, or in the form of an oral slow release lozenge to preventactivation of caspases and apoptotic cell death induced by thechemotherapeutic agent or by radiation. In the case of gastrointestinalmucositis, the caspase inhibitor can be applied in a form such that itis not absorbed systemically or in a form that coats the surface of thegastrointestinal tract, or a suppository formulation for the treatmentof gastrointestinal mucositis. In the case of proctitis, the caspaseinhibitor may be applied as part of an enema or suppository. In the caseof bladder mucositis, the caspase inhibitor may be applied though abladder catheter. For prevention of radiation or chemotherapy-inducedhair loss, the caspase inhibitor can be applied, for example, to thescalp in the form of a hair rinse, hair gel, shampoo or hairconditioner. Importantly, the caspase inhibitor can be applied prior tothe administration of the chemotherapeutic agent or radiation, thuspreventing the onset of the damaging effects of the chemotherapeuticagent or radiation to the normal cells.

[0139] The cell death inhibitors of the present invention may also beused to treat or prevent the inflammatory response in psoriasis orinflammatory bowel disease.

[0140] Compositions within the scope of this invention include allcompositions wherein the compounds of the present invention arecontained in an amount which is effective to achieve its intendedpurpose. While individual needs vary, determination of optimal ranges ofeffective amounts of each component is within the skill of the art.Typically, the compounds may be administered to mammals, e.g. humans,orally at a dose of 0.0025 to 50 mg/kg, or an equivalent amount of thepharmaceutically acceptable salt thereof, per day of the body weight ofthe mammal being treated for apoptosis-mediated disorders, e.g.,neuronal cell death, heart disease, retinal disorders, polycystic kidneydisease, immune system disorders and sepsis. Preferably, about 0.01 toabout 10 mg/kg is orally administered to treat or prevent suchdisorders. For intramuscular injection, the dose is generally aboutone-half of the oral dose. For example, for treatment or prevention ofneuronal cell death, a suitable intramuscular dose would be about 0.0025to about 25 mg/kg, and most preferably, from about 0.01 to about 5mg/kg.

[0141] The unit oral dose may comprise from about 0.01 to about 50 mg,preferably about 0.1 to about 10 mg of the compound. The unit dose maybe administered one or more times daily as one or more tablets eachcontaining from about 0.1 to about 10, conveniently about 0.25 to 50 mgof the compound or its solvates.

[0142] In a topical formulation, the compound may be present at aconcentration of about 0.01 to 100 mg per gram of carrier. In apreferred embodiment, the compound is present at a concentration ofabout 0.07-1.0 mg/ml, more preferably, about 0.1-0.5 mg/ml, mostpreferably, about 0.4 mg/ml.

[0143] In addition to administering the compound as a raw chemical, thecompounds of the invention may be administered as part of apharmaceutical preparation containing suitable pharmaceuticallyacceptable carriers comprising excipients and auxiliaries whichfacilitate processing of the compounds into preparations which can beused pharmaceutically. Preferably, the preparations, particularly thosepreparations which can be administered orally or topically and which canbe used for the preferred type of administration, such as tablets,dragees, slow release lozenges and capsules, mouth rinses and mouthwashes, gels, liquid suspensions, hair rinses, hair gels, shampoos andalso preparations which can be administered rectally, such assuppositories, as well as suitable solutions for administration byinjection, topically or orally, contain from about 0.01 to 99 percent,preferably from about 0.25 to 75 percent of active compound(s), togetherwith the excipient.

[0144] Also included within the scope of the present invention are thenon-toxic pharmaceutically acceptable salts of the compounds of thepresent invention. Acid addition salts are formed by mixing a solutionof the particular cell death inhibitors of the present invention with asolution of a pharmaceutically acceptable non-toxic acid such ashydrochloric acid, fumaric acid, maleic acid, succinic acid, aceticacid, citric acid, tartaric acid, carbonic acid, phosphoric acid, oxalicacid, and the like. Basic salts are formed by mixing a solution of theparticular cell death inhibitors of the present invention with asolution of a pharmaceutically acceptable non-toxic base such as sodiumhydroxide, potassium hydroxide, choline hydroxide, sodium carbonate Trisand the like.

[0145] The pharmaceutical compositions of the invention may beadministered to any animal which may experience the beneficial effectsof the compounds of the invention. Foremost among such animals aremammals, e.g., humans, although the invention is not intended to be solimited.

[0146] The caspase inhibitors and pharmaceutical compositions thereofmay be administered by any means that achieve their intended purpose.For example, administration may be by parenteral, subcutaneous,intravenous, intramuscular, intraperitoneal, transdermal, buccal,intrathecal, intracranial, intranasal or topical routes. Alternatively,or concurrently, administration may be by the oral route. The dosageadministered will be dependent upon the age, health, and weight of therecipient, kind of concurrent treatment, if any, frequency of treatment,and the nature of the effect desired. In general, the caspase inhibitorsare administered locally to the tissues that are to be protected fromapoptosis and separately from the chemotherapeutic agent. For example,cisplatin may be administered by i.v. injection to treat a cancer suchas brain, lung, breast, liver, kidney, pancreatic, ovarian, prostaticcancer, and the caspase inhibitor administered locally to treat,ameliorate, or prevent apototic cell death in the mouth orgastrointestinal tract, such as a mouth wash for the treatment of oralmucositis; and IV injectable aqueous solution for the treatment of bonemarrow cell death; and an oral formulation suitable for coating thegastrointestinal surfaces or an emema or suppository formulation for thetreatment of gastrointestinal mucositis including proctitis. The caspaseinhibitors may also be applied through a bladder catheter for thetreatment, amelioration or prevention of bladder mucositis.Alternatively or concurrently, the caspase inhibitors may be appliedtopically to the skin and/or scalp to treat, ameliorate or preventapoptotic cell death of hair and skin cells. In a further embodiment,the chemotherapeutic agent or radiation may be applied locally to treata localized cancer such as brain, lung, breast, liver, kidney,pancreatic, ovarian, prostatic cancer, and the caspase inhibitoradministered systemically, e.g. by i.v. injection, to treat, ameliorateor prevent apoptotic cell death of the gastrointestinal tract cells,mouth epithelial cells, bone marrow cells, skin cells and hair cells. Inthe case of oral mucositis in brain cancer treatment, for example, acaspase inhibitor that does not penetrate the blood-brain barrier can beapplied, for example, systemically by i.v. injection followed byirradiation of the brain tumor. This would protect the oral mucosa fromthe harmful effects of radiation but the caspase inhibitor would notprotect the brain tumor from the therapeutic effects of radiation.Importantly, the caspase inhibitor can be applied prior toadministration of the radiation, thus preventing the onset of thedamaging effects of the radiation to the normal mucosa cells.

[0147] The pharmaceutical preparations of the present invention aremanufactured in a manner which is itself known, for example, by means ofconventional mixing, granulating, dragee-making, dissolving, orlyophilizing processes. Thus, pharmaceutical preparations for oral usecan be obtained by combining the active compounds with solid excipients,optionally grinding the resulting mixture and processing the mixture ofgranules, after adding suitable auxiliaries, if desired or necessary, toobtain tablets or dragee cores.

[0148] Suitable excipients are, in particular, fillers such assaccharides, for example lactose or sucrose, mannitol or sorbitol,cellulose preparations and/or calcium phosphates, for example tricalciumphosphate or calcium hydrogen phosphate, as well as binders such asstarch paste, using, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, tragacanth, methyl cellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/orpolyvinyl pyrrolidone. If desired, disintegrating agents may be addedsuch as the above-mentioned starches and also carboxymethyl-starch,cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a saltthereof, such as sodium alginate. Auxiliaries are, above all,flow-regulating agents and lubricants, for example, silica, talc,stearic acid or salts thereof, such as magnesium stearate or calciumstearate, and/or polyethylene glycol. Dragee cores are provided withsuitable coatings which, if desired, are resistant to gastric juices.For this purpose, concentrated saccharide solutions may be used, whichmay optionally contain gum arabic, talc, polyvinyl pyrrolidone,polyethylene glycol and/or titanium dioxide, lacquer solutions andsuitable organic solvents or solvent mixtures. In order to producecoatings resistant to gastric juices, solutions of suitable cellulosepreparations such as acetylcellulose phthalate orhydroxypropymethyl-cellulose phthalate, are used. Dye stuffs or pigmentsmay be added to the tablets or dragee coatings, for example, foridentification or in order to characterize combinations of activecompound doses.

[0149] Other pharmaceutical preparations which can be used orallyinclude push-fit capsules made of gelatin, as well as soft, sealedcapsules made of gelatin and a plasticizer such as glycerol or sorbitol.The push-fit capsules can contain the active compounds in the form ofgranules which may be mixed with fillers such as lactose, binders suchas starches, and/or lubricants such as talc or magnesium stearate and,optionally, stabilizers. In soft capsules, the active compounds arepreferably dissolved or suspended in suitable liquids, such as fattyoils, or liquid paraffin. In addition, stabilizers may be added.

[0150] Possible pharmaceutical preparations which can be used rectallyinclude, for example, suppositories, which consist of a combination ofone or more of the active compounds with a suppository base. Suitablesuppository bases are, for example, natural or synthetic triglycerides,or paraffin hydrocarbons. In addition, it is also possible to usegelatin rectal capsules which consist of a combination of the activecompounds with a base. Possible base materials include, for example,liquid triglycerides, polyethylene glycols, or paraffin hydrocarbons.

[0151] Suitable formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form, forexample, water-soluble salts and alkaline solutions. In addition,suspensions of the active compounds as appropriate oily injectionsuspensions may be administered. Suitable lipophilic solvents orvehicles include fatty oils, for example, sesame oil, or synthetic fattyacid esters, for example, ethyl oleate or triglycerides or polyethyleneglycol-400 (the compounds are soluble in PEG-400). Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension include, for example, sodium carboxymethyl cellulose,sorbitol, and/or dextran. Optionally, the suspension may also containstabilizers.

[0152] In accordance with one aspect of the present invention, compoundsof the invention are employed in topical and parenteral formulations andare used for the treatment of skin damage, such as that caused byexposure to high levels of radiation, including ultraviolet radiation,heat or chemicals.

[0153] One or more additional substances which have therapeutic effectson the skin may also be incorporated in the compositions. Thus, thecomposition may also contain one or more compounds capable of increasingcyclic-AMP levels in the skin. Suitable compounds include adenosine or anucleic acid hydrolysate in an amount of about 0.1-1% and papaverine, inan amount of about 0.5-5%, both by weight based on the weight of thecomposition. Also suitable are β-adrenergic agonists such asisoproterenol, in an amount of about 0.1-2% or cyclic-AMP, in an amountof about 0.1-1%, again both by weight based on the weight of thecomposition. Other suitable types of additional active ingredients whichmay be incorporated in the compositions of this invention include anycompounds known to have a beneficial effect on skin. Such compoundsinclude retinoids such as Vitamin A, in an amount of about 0.003-0.3% byweight and chromanols such as Vitamin E or a derivative thereof in anamount of about 0.1-10% by weight, both based on the weight of thecomposition. Additionally, anti-inflammatory agents and keratoplasticagents may be incorporated in the cosmetic composition. A typicalanti-inflammatory agent is a corticosteroid such as hydrocortisone orits acetate in an amount of about 0.25-5% by weight, or a corticosteroidsuch as dexamethasone in an amount of about 0.025-0.5% by weight, bothbased on the weight of the composition. A typical keratoplastic agent iscoal tar in an amount of about 0.1-20% or anthralin in an amount ofabout 0.05-2% by weight, both based on the weight of the composition.

[0154] The topical compositions of this invention are formulatedpreferably as oils, creams, lotions, ointments and the like by choice ofappropriate carriers. Suitable carriers include vegetable or mineraloils, white petrolatum (white soft paraffin), branched chain fats oroils, animal fats and high molecular weight alcohol (greater than C₁₂).The preferred carriers are those in which the active ingredient issoluble. Emulsifiers, stabilizers, humectants and antioxidants may alsobe included as well as agents imparting color or fragrance, if desired.Additionally, transdermal penetration enhancers can be employed in thesetopical formulations. Examples of such enhancers can be found in U.S.Pat. Nos. 3,989,816 and 4,444,762.

[0155] Creams are preferably formulated from a mixture of mineral oil,self-emulsifying beeswax and water in which mixture the activeingredient, dissolved in a small amount of an oil such as almond oil, isadmixed. A typical example of such a cream is one which includes about40 parts water, about 20 parts beeswax, about 40 parts mineral oil andabout 1 part almond oil.

[0156] Ointments may be formulated by mixing a solution of the activeingredient in a vegetable oil such as almond oil with warm soft paraffinand allowing the mixture to cool. A typical example of such an ointmentis one which includes about 30% almond oil and about 70% white softparaffin by weight.

[0157] Lotions may be conveniently prepared by dissolving the activeingredient, in a suitable high molecular weight alcohol such aspropylene glycol or polyethylene glycol.

[0158] In addition, these compositions may include other medicinalagents, growth factors, wound sealants, carriers, etc., that are knownor apparent to those skilled in the art. The compositions of theinvention are administered to a warm-blooded animal, such as human,already suffering from a skin damage, such as a burn, in an amountsufficient to allow the healing process to proceed more quickly than ifthe host were not treated. Amounts effective for this use will depend onthe severity of the skin damage and the general state of health of thepatient being treated. Maintenance dosages over a prolonged period oftime may be adjusted as necessary. For veterinary uses, higher levelsmay be administered as necessary.

[0159] In the case of an animal suffering from decreased hair growth,the compositions of the invention are administered in an amountsufficient to increase the rate of hair growth. Amounts effective forthis use will depend on the extent of decreased hair growth, and thegeneral state of health of the patient being treated. Maintenancedosages over a prolonged period of time may be adjusted as necessary.For veterinary uses, higher levels may be administered as necessary.

[0160] When the compounds are to be administered to plants, they may beapplied to the leaves and/or stems and/or flowers of the plant, e.g. byspraying. The compounds may be spayed in particulate form or dissolvedor suspended in an appropriate carrier, e.g. in water or an oil-wateremulsion. The compounds may also be combined with the soil of the plant.In this embodiment, the compounds are taken up by the roots of theplant.

[0161] In a preferred embodiment, the caspase inhibitor is formulated aspart of a mouthwash for the treatment, amelioration or prevention oforal mucositis. Such mouthwashes are aqueous solutions of the caspaseinhibitor which may also contain alcohol, glycerin, synthetic sweetenersand surface-active, flavoring and coloring agents. They may also containanti-infective agents such as hexetidine and cetylpyridinium chloride.The mouthwashes may also contain topical anesthetics (e.g. benzocaine,cocaine, dyclonine hydrochloride, lidocaine, proparacaine hydrochlorideor teracaine hydrochloride), for example, for relieving pain ofradiation or chemotherapy-induced sores. The mouth washes may haveeither acidic or basic pH. See Remington's Pharmaceutical Sciences, A.R. Gennaro (ed.), Mack Publishing Company, pp. 1045, 1046, 1526 and 1965(1990).

[0162] In another preferred embodiment, the caspase inhibitor isformulated as an oral formulation which is capable of coating thegastrointestinal surfaces for the treatment, amelioration or preventionof gastrointestinal mucositis. Examples of gastrointestinal mucositisinclude esophageal mucositis, gastric mucositis, and intestinalmucositis. Such formulations may comprise gastric antacids such asaluminum carbonate, aluminum hydroxide gel, bismuth subnitrate, bismuthsubsalicylate, calcium carbonate, dihydroxyaluminum sodium carbonate,magaldrate, magnesium carbonate, magnesium hydroxide, magnesium oxide,sodium bicarbonate, milk of bismuth, dihydroxyaluminum aminoacetate,magnesium phosphate, magnesium trisilicate and mixtures thereof. Otheradditives include without limitation H₂-receptor antagonists,digestants, anti-emetics, adsorbants, and miscellaneous agents. SeeRemington's Pharmaceutical Sciences, A. R. Gennaro (ed.), MackPublishing Company, pp. 774-778 (1990).

[0163] Chemotherapy agents such as cisplatin and radiation therapy ofteninduce early and late onset emesis in the patient. Thus, in oneembodiment an antiemetic is coadminstered together with the caspaseinhibitor to avoid emesis and retain contact of the caspase inhibitorwith the gastrointestinal tract. Examples of such antiemetics includewithout limitation compounds that block the dopaminergic emeticreceptors such as metoclopramide and trimethobenzamide, andcannabinoids. Metoclopramide may be administered orally prior to and/orduring chemotherapy/radiation therapy/caspase inhibitor therapy toprevent the early emesis response and then later by intranasaladministration according to U.S. Pat. Nos. 5,760,086 and 4,536,386 toprevent delayed onset emesis. During the period afterchemotherapy/radiation therapy, both the caspase inhibitor and theantiemetic may be coadministered to treat, ameliorate or preventgastrointestinal mucositis.

[0164] In a further embodiment, the caspase inhibitor may be formulatedas an IV injectable solution for the treatment, amelioration orprevention of bone marrow cell death.

[0165] The compositions may be administered to a warm-blooded animal,such as human, already suffering from chemotherapy or radiationtherapy-induced non-cancer cell death, or, more preferably, before orduring therapy with chemotherapy or radiation.

[0166] The following examples are illustrative, but not limiting, of themethod and compositions of the present invention. Other suitablemodifications and adaptations of the variety of conditions andparameters normally encountered in clinical therapy and which areobvious to those skilled in the art are within the spirit and scope ofthe invention.

EXAMPLE 1 2-(Z-Amino)benzoyl-Asp-fmk

[0167] Step A. 2-Z-Aminobenzoic acid. To a solution of 2-aminobenzoicacid (0.30 g, 2.2 mmol) in pyridine (2 mL) was added benzylchloroformate (0.6 mL, 4.2 mmol) at 0° C. The mixture was then stirredat room temperature for 1 h, diluted with EtOAc (50 mL), washed with 2NHCl, water and brine, dried over Na₂SO₄ and concentrated in vacuo. Thecrude solid was washed with 4:1 hexane/EtOAc twice and dried in vacuo toyield the title compound as a white solid (270 mg, 1.0 mmol, 45%). ¹HNMR (DMSO-d₃): δ 11.49 (br s, 1H), 8.22 (d, J=7.5, 1 H), 7.95 (d, J=7.5,1H), 7.51 (t, J=7.5, 1H), 7.39-7.32 (m, 5 H), 7.05 (d, J=7.5, 1 H), 5.15(s, 2 H).

[0168] Step B. tert-Butyl5-fluoro-3-[2-Z-aminobenzoylamido]-4-hydroxypentanoate. A mixture of2-Z-aminobenzoic acid (90 mg, 0.33 mmol),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 61mg, 0.40 mmol), 1-hydroxybenzotriazole hydrate (HOBT, 73 mg, 0.38 mmol),dimethylaminopyridine (DMAP, 22 mg, 0.18 mmol) and tert-butyl3-amino-5-fluoro-4-hydroxypentanoate (79 mg, 0.38 mmol) in THF (10 mL)was stirred at room temperature for 20 h, diluted with 1:1 hexane/EtOAc(75 mL), washed with water, 2N HCl, water, 2N NaOH and brine, dried overNa₂SO₄ and cocentrated in vacuo. The residue was purified bychromatography (3:2 Hexane/EtOAc) to yield the title compound as ayellow hydroscopic solid (52 mg, 0.11 mmol, 33%).

[0169] Step C. 2-(Z-Amino)benzoyl-Asp(OBu-t)-fmk. A mixture ofperiodinane (0.41 g, 0.97 mmol) and tert-butyl5-fluoro-3-[2-Z-aminobenzoylamido]-4-hydroxypentanoate (52 mg, 0.11mmol) in dichloromethane (15 mL) was refluxed for 20 h, cooled to roomtemperature, and 25 mL of saturated sodium bicarbonate aqueous solutioncontaining 1.0 g of Na₂S₂O₃ was added. The resulting mixture was stirredfor 2 h, diluted with 1:1 hexane/EtOAc (75 mL), washed with water andbrine, dried over Na₂SO₄ and concentrated in vacuo. The residue waspurified by chromatography (3:2 hexane/EtOAc) to yield the titlecompound as a hydroscopic yellow solid (45 mg, 0.10 mmol, 91%). ¹H NMR(CDCl₃): δ 10.48 (s, 1H), 8.42 (d, J=8.7, 1H), 7.51-7.33 (m, 7H), 7.07(m, 1 H), 5.30-4.98 (m, 1H), 5.21 (s, 2H), 3.11-2.83 (m, 2 H), 1.44 (s,9 H).

[0170] Step D. 2-(Z-Amino)benzoyl-Asp-fmk. To a solution of2-(Z-Amino)benzoyl-Asp(OBu-t)-fmk (45 mg, 0.10 mmol) in 5 mL of CH₂Cl₂was added 1 mL of TFA. The resulting solution was allowed to stir at rtfor 1 hr, diluted with EtOAc (75 ml), washed with water, aqueous Na₂HPO₄to pH 5 and then brine, dried over Na₂SO₄ and concentrated in vacuo togive the title compound as a white solid (15 mg, 0.037 mmol, 38%). ¹HNMR (DMSO-d₆): δ 10.63 (s, 1H), 9.08 (s, 1 H), 8.14 (d, J=7.8, 1 H),7.76 (d, J=7.8, 1 H), 7.54 (t, J=7.8, 1 H), 7.41-7.35 (m, 5 H), 7.15 (t,J=7.8, 1H), 5.16 (s, 2H), 5.26-4.95 (m, 2H), 4.83 (m, 1H), 3.00-2.64 (m,2 H).

EXAMPLE 2 2-(Z-Amino)-6-methylbenzoyl-Asp-fmk

[0171] The title compound was prepared in four-steps as described inExample 1 from 2-amino-6-methylbenzoic acid. ¹H NMR (DMSO-d₆): δ 8.85(s, 1H), 8.68 (s, 1H), 7.49-7.01 (m, 8H), 5.12 (s, 2H), 4.82 (m, 1H),5.26-4.95 (m, 2H), 3.00-2.64 (m. 2H), 2.26 (s, 3H).

EXAMPLE 3 2-(Z-Amino)-5-methylbenzoyl-Asp-fmk

[0172] The title compound was prepared in four-steps as described inExample 1 from 2-amino-5-methylbenzoic acid. ¹H NMR (DMSO-d₆): δ 10.48(s, 1H), 9.10 (d, J=9, 1H), 8.00 (d, J=8.7, 1H), 7.89 (s, 1H), 7.58 (s,1H), 7.41-7.34 (m, 5H), 5.14 (s, 2H), 4.83 (m, 1H), 5.39-4.41 (m, 2H),2.94-2.80 (m. 2H), 2.30 (s, 3H).

EXAMPLE 4 2-(Z-Amino)-3-methylbenzoyl-Asp-fmk

[0173] The title compound was prepared in four-steps as described inExample 1 from 2-amino-3-methylbenzoic acid. ¹H NMR (DMSO-d₆): δ 9.05(s, 1H), 8.73 (s, 1H), 7.38-7.20 (m, 8H), 5.08 (s, 2H), 4.72 (m, 1H),5.32 (bs, 2H), 2.81-2.66 (m. 2H), 2.21 (s, 3H).

EXAMPLE 5 2-(Z-Amino)-3-methoxybenzoyl-Asp-fmk

[0174] The title compound was prepared in four-steps as described inExample 1 from 2-amino-3-methoxybenzoic acid. ¹H NMR (DMSO-d₆): δ 8.73(bs, 1H), 8.64 (bs, 1H), 7.57-7.06 (m, 9H), 5.06 (s, 2H), 4.72 (bs, 1H),5.26-4.97 (m, 2H), 3.78 (s, 3H), 2.78-2.66 (m. 2H).

EXAMPLE 6 2-(Z-Amino)-5-fluorobenzoyl-Asp-fmk

[0175] The title compound was prepared in four-steps as described inExample 1 from 2-amino-5-fluorobenzoic acid. ¹H NMR (DMSO-d₆): δ 10.40(bs, 1H), 9.13 (bs, 1H), 8.07 (q, J=5.1, 1H), 7.61 (d, J=6.6, 1H),7.46-7.33 (m, 6H), 5.15 (s, 2H), 4.81 (bs, 1H), 2.84-2.72 (m. 2H).

EXAMPLE 7 cis-2-(Z-Amino)cyclohexanecarboxyl-Asp-fmk

[0176] The title compound was prepared in four-steps as described inExample 1 from cis-2-aminocyclohexanecarboxylic acid. ¹H NMR (DMSO-d₆):δ 8.28 (bs, 1H), 7.39-7.09 (m, 5H), 4.98 (s, 2H), 4.52-4.45 (m, 1H),3.99 (bs, 1H), 2.62-2.53 (m, 4H), 1.77-1.23 (m, 8H).

EXAMPLE 8 2-(Z-Amino)-3,5-dimethylbenzoyl-Asp-fmk

[0177] The title compound was prepared in four-steps as described inExample 1 from 2-amino-3,5-dimethylbenzoic acid. ¹H NMR (DMSO-d₆): δ8.05 (s, 1H), 7.42-7.17 (m, 8H), 5.15 (s, 2H), 5.19-5.03 (m, 2H), 4.87(m, 1H), 2.30 (s, 3H), 2.26 (s, 3H).

EXAMPLE 9 2-(Z-Amino)-5-chlorobenzoyl-Asp-fmk

[0178] The title compound was prepared in four-steps as described inExample 1 from 2-amino-5-chlorobenzoic acid. ¹H NMR (DMSO-d₆): δ 10.56(s, 1H), 9.19 (s, 1H), 8.15 (d, J=9.0, 1H), 7.84 (s, 1H), 7.61 (d,J=9.0, 1H), 7.41-7.37 (m, 5H), 5.16 (s, 2H), 4.81 (m, 1H), 5.41-4.80 (m,2H), 2.84-2.73 (m, 2H).

EXAMPLE 10 2-(Z-Amino)-6-chlorobenzoyl-Asp-fmk

[0179] The title compound was prepared in four-steps as described inExample 1 from 2-amino-6-chlorobenzoic acid. ¹H NMR (DMSO-d₆): δ 9.17(d, J=4.2, 1H), 8.95 (s, 1H), 7.74-7.24 (m, 8H), 5.50-5.21 (m, 2H), 5.15(s, 2H), 4.85-4.78 (m, 1H), 2.98-2.65 (m, 2H).

EXAMPLE 11 2-(Z-Amino)-4-chlorobenzoyl-Asp-fmk

[0180] 5 The title compound was prepared in four-steps as described inExample 1 from 2-amino-3,5-dimethylbenzoic acid. ¹H NMR (DMSO-d₆): δ10.84 (s, 1H), 9.19 (s, 1H), 8.24 (s, 1H), 7.81 (d, J=8.4, 1H),7.42-7.24 (m, 6H), 5.18 (s, 2H), 5.25-5.20 (m, 2H), 4.82 (m, 1H),2.94-2.63 (m, 2H).

EXAMPLE 12 3-(Z-Amino)thiophene-2-carboxyl-Asp-fmk

[0181] Step A. 3-(Z-Amino)thiophene-2-carboxylic acid. The mixture ofmethyl 3-aminothiophene-2-carboxylate (0.2 g, 1.27 mmol) in 2N NaOH (10mL) was heated at 90° C. for 15 min, then cooled to 0° C. To theresulting solution was added benzyl chloroformate (1.5 mL, 10.5 mmol)and THF (10 mL). The mixture was then stirred at room temperature for 1h, washed with 3:1 hexane:ethyl acetate (2×15 mL). The aqueous phase wasacidified with 2N HCl to pH˜1-2, extracted with ethyl acetate (3×15 mL),washed with water and brine, dried over Na₂SO₄ and concentrated in vacuoto yield the title compound as a white solid (70 mg).

[0182] The title compound was then prepared in three steps as describedin Example 1 (B-D). ¹H NMR (DMSO-d₆): δ 10.43 (s, 1H), 8.74 (s, 1H),7.81 (d, J=5.4, 1H), 7.73 (d, J=5.4, 1H), 7.44-7.35 (m, 5H), 5.18 (s,2H), 5.32-5.04 (m, 2H), 4.79 (m, 1H), 2.88-2.67 (m, 2H).

EXAMPLE 13 3-(Methoxycarbonylamino)thiophene-2-carboxyl-Asp-fmk

[0183] The title compound was prepared in four-steps as described inExample 12 from methyl 3-aminothiophene-2-carboxylate and methylchloroformate. ¹H NMR (DMSO-d₆): δ 10.34 (s, 1H), 8.70 (s, 1H), 7.79 (d,J=5.7, 1H), 7.72 (d, J=5.7, 1H), 5.31-4.80 (m, 3H), 3.70 (s, 3H),2.96-2.73 (m, 2H).

EXAMPLE 14 Cis-2-(Z-Amino)cyclopentanecarboxyl-Asp-fmk

[0184] The title compound was prepared in four-steps as described inExample 1 from cis-2-aminocyclopentanecarboxylic acid. ¹H NMR (DMSO-d₆):δ 8.35 (s, 1H), 7.34-7.28 (m, 5H), 7.09 (m, 1H), 5.13-4.50 (m, 5H), 4.11(m, 1H), 2.81 (m, 1H), 2.73-2.51 (m, 2H), 1.91-1.40 (m, 6H).

EXAMPLE 15 Trans-2-(Z-Amino)cyclohexanecarboxyl-Asp-fmk

[0185] The title compound was prepared in four-steps as described inExample 1 from trans-2-aminocyclohexanecarboxylic acid. ¹H NMR(DMSO-d₆): δ 12.48 (s, 1H), 8.26-8.15 (m, 1H), 7.38-7.17 (m, 5H),5.18-4.47 (m, 5H), 2.67-2.50 (m, 2H), 2.19 (m, 1H), 1.83-1.06 (m, 10H).

EXAMPLE 16 Z-Glu-(2-aminobenzoyl)-Asp-fmk

[0186] Step A. Z-Glu(OBu-t)-2-aminobenzoic acid. To a solution ofZ-Glu(OBu-t)OH (272 mg, 0.81 mmol) in THF (5 mL) was addedN-methylmorpholine (110 μL, 1.1 mmol) at −45° C., followed by isobutylchloroformate (105 μL, 0.81 mmol). The mixture was stirred at −45° C.for 30 min. and a solution of anthranllic acid (127 mg, 0.93 mmol) inTHF (5 mL) was added, followed by more N-methylmorpholine (200 μL, 1.82mmol). The resulting mixture was stirred overnight and the cooling bathwas allowed to slowly warm to room temperature. After diluted with EtOAc(100 mL), the mixture was washed with 2N HCl, water, saturated NaHCO₃,water, 2N HCl, water and brine, dried over Na₂SO₄ and concentrated invacuo to yield the product as a highly hydroscopic white solid (330 mg,0.72 mmol, 89%). ¹H NMR (DMSO-d₆): δ 11.79 (s, 1H), 8.59 (d, J=8.6, 1H),8.00 (t, J=6.0, 1H), 7.60 (t, J=8.6, 1H), 7.39-7.31 (m, 5H), 7.17 (t,J=7.8, 1H), 5.16-4.99 (m, 2H), 4.08 (m, 1H), 2.33 (m, 2H), 2.08-1.75 (m,2H), 1.38 (s, 9H).

[0187] Step B. tert-Butyl5-fluoro-3-[Z-Glu(OBu-t)-(2-aminobenzoyl)amido]-4-hydroxypentanoate. Amixture of Z-Glu(OBu-t)-2-aminobenzoic acid (330 mg, 0.72 mmol), EDCI(129 mg, 0.67 mmol), HOBT (104 mg, 0.68 mmol), DMAP (46 mg, 0.38 mmol)and tert-butyl 3-amino-5-fluoro4-hydroxypentanoate (136 mg, 0.66 mmol)in THF (6 mL) was stirred at room temperature for 20 h. After dilutedwith 1:1 hexane/EtOAc (75 mL), the mixture was washed with water, 2NHCl, water, 2N NaOH and brine, dried over Na₂SO₄ and concentrated invacuo. The residue was purified by chromatography (3:1 then 3:2hexane/EtOAc) to yield the title compound as a white solid (45 mg, 0.068mmol, 10%).

[0188] Step C. Z-Glu(OBu-t)-[2-aminobenzoyl]-Asp(OBu-t)-fmk. The titlecompound was synthesized by a similar procedure as described in Step C,Example 1 in 58% yield.

[0189] Step D. Z-Glu-[2-aminobenzoyl]-Asp-fmk. The title compound wassynthesized by a similar procedure as described in Step D, Example 1 in14% yield. ¹H NMR (DMSO-d₆): δ 11.46 (s, 1H), 9.18 (s, 1H), 8.57-7.20(m, 6H), 5.36-4.84 (m, 5H), 4.04 (br s, 1H), 2.95-1.81 (m, 6H).

EXAMPLE 17 Z-Val-(2-Aminobenzoyl)-Asp-fmk

[0190] The title compound was synthesized as described in Example 16from Z-Val. ¹H NMR (DMSO-d₆): δ 11.34-11.25 (m, 1H), 9.17-7.17 (m, 11H),5.42-4.30 (m, 5H), 3.95-3.75 (m, 1H), 2.95-2.57 (m, 2H), 1.92 (m, 1H),0.91-0.84 (m, 6H).

EXAMPLE 18 2-(Z-Amino)benzoyl-Asp-DCB-methylketone

[0191] Step A. Z-Asp(OBu-t)-DCB-methylketone To a solution ofZ-Asp(OBu-t)-bromomethylketone (500 mg, 1.24 mmol) in DMF (10 ml) wasadded potassium fluoride (320 mg, 5.50 mmol), and 2,6-dichlorobenzoicacid (348 mg, 1.82 mmol). The mixture was stirred at room temperaturefor 12 h, and then was diluted with 25 ml of ethyl acetate, washed withaqueous NH₄Cl and brine, dried over Na₂SO₄ and concentrated in vacuo.The title compound was obtained as white solid (0.78 g, 2.62 mmol, 69%).¹H NMR (CDCl₃): 7.34 (m, 8H), 5.96 (d, J=8.7, 1H), 5.21 (d, J=6.6, 2H),5.16 (s, 2H), 4.70 (m, 1H), 2.88 (m, 2H), 1.27 (s, 9H).

[0192] Step B. Asp(OBu-t)-DCB-methylketone-HCl To a solution ofZ-Asp(OBu-t)-DCB-methylketone (572 mg, 1.14 mmol) in ethanol (15 ml) wasadded Pd/C (50 mg) and 6N HCl (0.2 ml). The mixture was stirred at roomtemperature under H₂ atmosphere (1 atm) for 12 h, then it was filteredand concentrated. The title compound was obtained as pale white solid(416 mg, 1.04 mmol, 90%). ¹H NMR (CDCl₃): 7.27 (m, 3H), 5.28 (m, 2H),4.94 (m, 1H), 3.27 (m, 2H), 1.42 (s, 9H).

[0193] Step C. 2-(Z-Amino)benzoyl-Asp(OBu-t)-DCB-methylketone To asolution of 2-(Z-amino)benzoic acid (140 mg, 0.52 mmol) in THF (5 mL)was added N-methylmorpholine (65 μL, 0.59 mmol), followed by2-methylpropyl chloroformate (70 μL, 0.54 mmol) at −45° C. After 30min., a solution of Asp(OBu-t)-DCB-methylketone-HCl (121 mg, 0.27 mmol)in THF (5 mL) was added to the solution. The resulting mixture wasfurther stirred overnight and the cooling bath was allowed to slowlywarm to room temperature. It was then diluted with 1:1 hexane/EtOAc (100mL), washed with water, 2N NaOH and brine, dried over Na₂SO₄ andconcentrated in vacuo. The residue was purified by chromatography (3:1hexane/EtOAc) to yield the title compound as a white solid (365 mg, 0.05mmol, 19%). ¹H NMR (CDCl₃): 10.90 (s, 1H), 8.49 (d, J=7.5, 1H), 7.87(dd, J=8.1, 1.8, 1H), 7.56-7.32 (m, 9H), 7.08 (t, J=6.9, 1H), 5.23 (s,2H), 5.28 (m, 1H), 4.90 (d, J=1.8, 2H), 3.16-2.91 (m, 1H), 1.43 (m, 9H).

[0194] Step D. 2-(Z-Amino)benzoyl-Asp-DCB-methylketone A solution of2-(Z-amino)benzoyl-Asp(OBu-t)-DCB-methylketone (35 mg) and TFA (1 mL) inmethylenechloride (3 mL) was stirred at room temperature for 2 h. Themixture was diluted with EtOAc (70 mL), washed with saturated Na₂HPO₄ topH˜5, and further washed with water, and brine, dried over Na₂SO₄ andconcentrated in vacuo to yield the title compound as a white solid (10mg, 0.016 mmol, 33%). ¹H NMR (CDCl₃): 10.94 (s, 1H), 8.49 (d, J=8.4,1H), 7.87 (dd, J=8.1, 1.5, 1H), 7.53 (m, 1H), 7.45-7.33 (m, 8H), 7.07(m, 1H), 5.22 (s, 2H), 5.10-5.07 (m, 1H), 4.90 (m, 2H), 3.11 (dd, J=8.4,19.0, 1H), 2.95 (dd, J=1.8, 19.0, 1H)).

EXAMPLE 19 Methoxycarbonyl-Val-(2-aminobenzoyl)-Asp-fmk

[0195] Step A. tert-Butyl5-fluoro-3-(2-aminobenzoylamido)-4-hydroxypentanoate.HCl. A mixture oftert-butyl 5-fluoro-3-(2-Z-aminobenzoylamido)-4-hydroxypentanoate (80mg, 0.174 mmol), Pd-C (23 mg) and 6N HCl (0.087 mL) in ethanol (5 mL)was stirred under hydrogen atmosphere at room temperature for 2 h. Themixture was filtered and the solvent was evaporated to yield the titleproduct. It was used in next step without further purification.

[0196] Step B. tert-Butyl5-fluoro-3-(methoxycarbonyl-Val-2-aminobenzoylamido)-4-hydroxypentanoate.To a solution of methoxycarbonyl-Val-OH (31 mg, 0.17 mmol) in THF (5 mL)was added N-methylmorpholine (38 μL, 0.34 μmmol) at −45° C., followed byisobutyl chloroformate (45 μL, 0.034 mmol). The mixture was stirred at−45° C. for 30 min and a solution of tert-butyl5-fluoro-3-(2-aminobenzoylamido)-4-hydroxypentanoate.HCl in THF (5 mL)was added, followed by more N-methylmorpholine (50 μL, 0.45 mmol). Theresulting mixture was stirred overnight and the cooling bath was allowedto slowly warm to room temperature. After dilution with ethyl acetate(50 mL), the mixture was washed with water and brine, dried over Na₂SO₄and concentrated in vacuo. The residue was purified by chromatography(3:2 hexane:ethyl acetate) to yield the title compound as a whitehydroscopic solid (20 mg, 0.041 mmol, 24%). ¹H NMR (CDCl₃): δ11.36-11.24 (m, 1H), 8.54 (d, J=8.7, 1H), 7.55-7.05 (m, 4H), 5.40 (m,1H), 4.89-3.85 (m, 6H), 3.71 (d, J=1.8, 3H), 2.84-2.61 (m, 2H), 2.29 (m,1H), 1.46-1.44 (m, 9H), 1.05-0.98 (m, 6H).

[0197] Step C, D. Methoxycarbonyl-Val-(2-aminobenzoyl)-Asp-fmk. Thetitle compound was synthesized with a similar procedure as described inStep C and D of Example 1. ¹H NMR (DMSO-d₆): δ 12.52 (s, 1H), 11.27 (d,J=6.0, 1H), 9.18 (m, 1H), 7.88-7.54 (m, 4H), 7.20 (t, J=7.5, 1H),5.39-4.44 (m, 3H), 3.83 (m, 1H), 3.57 (d, J=2.4, 3H), 2.96-2.65 (m, 2H),2.18 (m, 1H), 0.92 (t, J=6.3, 6H).

EXAMPLE 20 Enzyme Activity

[0198] The activity of 2-(Z-amino)benzoyl-Asp-fmk as an inhibitor ofcaspase-3 was measured in a fluorometric enzyme assay. Enzyme activitywas measured using synthetic peptide substrates attached to afluorogenic leaving group. Cleavage of the synthetic substrate by theenzyme results in a fluorescent signal which is read in aspectrofluorometer or in a fluorometric microtiter plate reader.

[0199] 12 concentrations of the testing compound ranged from 30 pM to 10μM were tested in the enzyme assay. The enzyme reaction was conducted inthe presence of 2 ng rCaspase 3 (purchased from PharMingen, a Bectondivision company, San Diego, Calif.), various concentrations of testingcompound, 10 μM caspase 3 substrate Ac-DEVD-AMC (purchased from QualityControlled Biochemicals, Inc. Hopkinton, Mass.) and caspase buffer (20mM PIPES, 100 mM NaCl, 10 mM DTT, 1 mM EDTA, 0.1% CHAPS and 10% sucrose,pH 7.2) in a total volume of 100 μL. The enzyme reaction was carried outin a 96-well plate and incubated at 37° C. for 30 minutes. The plate wasthen read with a fluorescence plate reader (EG&G WALLAG 1420-002) usingexcitation filter at 355 nm/emission filter at 460 nm. The data wasanalyzed using GraphPrism software to give an IC₅₀ value of 0.2 μM.

[0200] Having now fully described this invention, it will be understoodby those of ordinary skill in the art that the same can be performedwithin a wide and equivalent range of conditions, formulations and otherparameters without affecting the scope of the invention or anyembodiment thereof. All patents, patent applications and publicationscited herein are fully incorporated by reference herein in theirentirety.

1 1 1 4 PRT Homo sapiens MOD RES (1)..(1) N-terminal acetyl 1 Asp GluVal Asp 1

What is claimed is:
 1. A compound having the Formulae I or II or III:

or a pharmaceutically acceptable salt or prodrug thereof, wherein: R₁ isan optionally substituted alkyl or hydrogen; R₃ is an N-protectinggroup; R₂ is hydrogen or optionally substituted alkyl; Q is anoptionally substituted saturated or partially saturated carbocycle orheterocycle; X is a peptide of 1-4 amino acids or a bond; Y is a peptideof 1-4 amino acids or a bond; A is CR₆ or nitrogen; B is CR₇ ornitrogen; C is CR₈ or nitrogen; D is CR₉ or nitrogen; provided that notmore than two of A, B, C or D is nitrogen; and R₆-R₉ independently arehydrogen, halo, C₁-C₆ haloalkyl, C₆-C₁₀ aryl, C₄-C₇ cycloalkyl, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₆-C₁₀ aryl(C₁-C₆)alkyl, C₆-C₁₀aryl(C₂-C₆)alkenyl, C₆-C₁₀ aryl(C₂-C₆)alkynyl, C₁-C₆ hydroxyalkyl,nitro, amino, cyano, C₁-C₆ acylamino, hydroxy, C₁-C₆ acyloxy, C₁-C₆alkoxy, alkylthio, or carboxy; or one of R₆ and R₇, or R₇ and R₈, or R₈and R₉ are taken together with the carbon atoms to which they areattached to form a carbocycle or heterocycle; E is C₁₄, nitrogen, oxygenor sulfur; F is C₁₅, nitrogen, oxygen or sulfur; G is C₁₆, nitrogen,oxygen or sulfur; provided that only one of E, F, G is nitrogen, oxygenor sulfur and R₁₄-R₁₆ are independently hydrogen, halo, C₁-C₆ haloalkyl,C₆-C₁₀ aryl, C₄-C₇ cycloalkyl, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₆-C₁₀ aryl(C₁-C₆)alkyl, C₆-C₁₀ aryl(C₂-C₆)alkenyl, C₆-C₁₀aryl(C₂-C₆)alkynyl, C₁-C₆ hydroxyalkyl, nitro, amino, cyano, C₁-C₆acylamino, hydroxy, C₁-C₆ acyloxy, C₁-C₆ alkoxy, alkylthio, or carboxy;or one of R₁₄ and R₁₅, or R₁₅ and R₁₆, are taken together with thecarbon atoms to which they are attached to form a carbocycle orheterocycle.
 2. A compound according to claim 1, wherein R₃ ist-butyloxycarbonyl, acetyl or benzyloxycarbonyl.
 3. A compound accordingto claim 1, wherein R₁ is H, Me, Et or acetoxymethyl.
 4. A compoundaccording to claim 1, wherein R₂ is hydrogen, fluoromethyl,acyloxymethyl, arylacyloxymethyl or aminomethyl.
 5. A compound accordingto claim 1, wherein X is a bond.
 6. A compound according to claim 1,wherein A, B, C and D are CH.
 7. A compound according to claim 1,wherein A is nitrogen, and B, C and D are CH.
 8. A compound according toclaim 1, wherein G is sulfur, and E and F are CH.
 9. A compoundaccording to claim 1, wherein Q is cyclohexyl or cyclopentyl.
 10. Acompound according to claim 1, wherein said compound has the Formula IV:

or a pharmaceutically acceptable salt or prodrug thereof, wherein R₂ ishydrogen or optionally substituted alkyl, wherein the substituent ishalo, hydroxy, alkoxy, aryloxy, alkylthio, arylthio, amino, acyloxy, orarylacyloxy; R₆-R₉ independently are hydrogen, halo, C₁-C₆ haloalkyl,C₆-C₁₀ aryl, C₄-C₇ cycloalkyl, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₆-C₁₀ aryl(C₁-C₆)alkyl, C₆-C₁₀ aryl(C₂-C₆)alkenyl, C₆-C₁₀aryl(C₂-C₆)alkynyl, C₁-C₆ hydroxyalkyl, nitro, amino, cyano, C₁-C₆acylamino, hydroxy, C₁-C₆ acyloxy, C₁-C₆ alkoxy, alkylthio, or carboxy;or one of R₆ and R₇, or R₇ and R₈, or R₈ and R₉ are taken together withthe carbon atoms to which they are attached to form a carbocycle orheterocycle, selected from the group consisting of —OCH₂O—, —OCF₂O—,—(CH₂)₃—, —(CH₂)₄—, —OCH₂CH₂O—, —CH₂N(R₁₃)CH₂—, —CH₂CH₂N(R₁₃)CH₂—,—CH₂N(R₁₃)CH₂CH₂— and —CH═CH—CH═CH—; wherein R₁₃ is hydrogen, alkyl orcycloalkyl; R₁₀ is hydrogen, C₁-C₆ alkoxy, C₁-C₆ alkyl, C₁-C₆ haloalkyl,C₆-C₁₀ aryl, C₄-C₇ cycloalkyl, C₆-C₁₀ aryl(C₁-C₆)alkyl, benzyloxy,substituted benzyloxy, or NR₁₁R₁₂; wherein R₁₁ and R₁₂ independently arehydrogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₆-C₁₀ aryl, C₄-C₇ cycloalkyl,C₆-C₁₀ aryl(C₁-C₆)alkyl, or R₁₁ and R₁₂ are combined to form aheterocyclic ring system selected from the group consisting ofpyrrolidine, piperidine, piperazine, and morpholine.
 11. A compoundaccording to claim 10, wherein R₂ is hydrogen, fluoromethyl,acyloxymethyl, arylacyloxymethyl or aminomethyl.
 12. A compoundaccording to claim 10, wherein R₁₀ is benzyloxy.
 13. A compoundaccording to claim 10, wherein R₁ is H, Me or acetoxymethyl.
 14. Acompound according to claim 10, wherein X is a peptide of 1-2 aminoacids or a bond.
 15. A compound according to claim 1, wherein saidcompound is selected from the group consisting of:2-(Z-amino)benzoyl-Asp-fmk, 2-(Z-amino)-3-methylbenzoyl-Asp-fmk,2-(Z-amino)-3,5-dimethylbenzoyl-Asp-fmk,2-(Z-amino)-4-chlorobenzoyl-Asp-fmk,2-(Z-amino)-5-chlorobenzoyl-Asp-fmk,2-(Z-amino)-5-fluorobenzoyl-Asp-fmk,2-(Z-amino)-6-fluorobenzoyl-Asp-fmk,cis-2-(Z-amino)-cyclohexanecarboxy-Asp-fmk,2-(Z-amino)-5-methylbenzoyl-Asp-fmk,2-(Z-amino)-6-methylbenzoyl-Asp-fmk,2-(Z-amino)-6-chlorobenzoyl-Asp-fmk,2-(Z-amino)-3-methoxybenzoyl-Asp-fmk,3-(Z-amino)thiophene-2-carboxyl-Asp-fmk,3-(methoxycarbonylamino)thiophene-2-carboxyl-Asp-fmk,cis-2-(Z-amino)cyclopentanecarboxyl-Asp-fmk,trans-2-(Z-amino)cyclopentanecarboxyl-Asp-fmk,2-(Z-amino)benzoyl-Asp-DCB-methylketone,methoxycarbonyl-Val-(2-aminobenzoyl)-Asp-fmk,Z-Glu-(2-aminobenzoyl)-Asp-fmk, and Z-Val-(2-aminobenzoyl)-Asp-fmk. 16.A pharmaceutical composition, comprising a compound of claim 1, and apharmaceutically acceptable carrier.
 17. A method of inhibiting celldeath of a cell or tissue, comprising contacting said cell or tissuewith an effective amount of a compound of claim
 1. 18. A method oftreating or ameliorating cell death in the central or peripheral nervoussystem, retinal neurons, cardiac muscle or immune system cells of ananimal, comprising administering to the animal in need of such treatmentor ameliorating an effective amount of a compound of claim1.
 19. Themethod of claim 18, wherein said cell death is in the central orperipheral nervous system, and is due to one of: (a) a condition ofischemia and excitotoxicity selected from the group consisting of focalischemia due to stroke and global ischemia due to cardiac arrest; (b)traumatic injury; (c) viral infection; (d) radiation-induced nerve celldeath; (e) a neurodegenerative disorder selected from the groupconsisting of Alzheimer's disease, Parkinson's Disease, a prion disease,multiple sclerosis, amyotrophic lateral sclerosis, and spinobulbaratrophy; (f) spinal cord injury; or (g) acute bacterial meningitis. 20.The method of claim 18, wherein said cell death is in the central orperipheral nervous system, and is due to expansion of trinucleotiderepeats of specific genes.
 21. The method of claim 18, wherein said celldeath is due to Huntington's Disease.
 22. The method of claim 18,wherein said cell death is in cardiac muscle tissue, and is due tomyocardial infarction, congestive heart failure, cardiomyopathy or viralinfection of the heart.
 23. The method of claim 18, wherein said celldeath is in retinal neurons and is due to increased intraocularpressure, age-related macular degeneration or retinitis pigmentosa. 24.The method of claim 18, wherein said cell death is in the immune system,and is due to an immune deficiency disorder selected from the groupconsisting of acquired immune deficiency syndrome, severe combinedimmune deficiency syndrome and radiation-induced immune suppression. 25.The method of claim 18, wherein said cell death is due to an autoimmunedisorder selected from the group consisting of lupus erythematosus,rheumatoid arthritis and type I diabetes.
 26. The method of claim 18,wherein said cell death is due to type I diabetes.
 27. A method oftreating or preventing polycystic kidney disease, renal amyloidosis,acute renal failure, cyclosporine A induced tubular epithelial celldeath, hypoxia-induced necrosis of renal proximal tubules, HIV-inducednephropathy or anemia/erythropoiesis in an animal, comprisingadministering to the animal in need of such treatment an effectiveamount of a compound of claim
 1. 28. A method of protecting a mammalianorgan or tissue from cell death due to deprivation of normal bloodsupply, comprising contacting said organ or tissue with an effectiveamount of a compound of claim
 1. 29. The method of claim 28, whereinsaid organ or tissue is present in a storage medium prior to transplantinto a mammal.
 30. The method of claim 28, wherein said contactingcomprises infusion of said compound into the organ or tissue, or bathingof said organ or tissue in a storage medium which comprises saidcompound.
 31. A method of reducing or preventing cell death in a donororgan or tissue after it has been transplanted into a host due to theeffects of reperfusion injury or due to the effects of host immunecells, comprising administering to said host in need thereof aneffective amount of a compound of claim
 1. 32. A method of reducing orpreventing the death of mammalian sperm or eggs used in in vitrofertilization procedures, comprising contacting said sperm or egg withan effective amount of a compound of claim
 1. 33. A method of extendingthe lifespan of a mammalian or yeast cell line, comprising contactingsaid cell line with a compound of claim
 1. 34. The method of claim 33,wherein said contacting comprises including said compound in a cellgrowth medium.
 35. A method of treating or ameliorating hair loss orpremature graying of the hair in a mammal, comprising contacting thehair or hair follicles of the mammal in need thereof with a compound ofclaim
 1. 36. The method of claim 35, wherein hair loss is treated, andsaid hair loss is due to male-pattern baldness, radiation, chemotherapyor emotional stress.
 37. A method of treating or ameliorating skindamage of a mammal due to exposure to high levels of radiation, heat orchemicals, comprising applying to the skin of the mammal in need thereofwith a compound of claim
 1. 38. The method of claim 37, wherein saidcompound is applied as part of an ointment.
 39. The method of claim 37,wherein said skin damage is due to acute over-exposure to the sun, andwherein said treating reduces blistering and peeling of the skin.
 40. Amethod of treating or ameliorating sepsis or multi-organ failure in ananimal, comprising administering to the animal in need thereof aneffective amount of a compound of claim
 1. 41. A method of treating orameliorating hepatitis in an animal, comprising administering to theanimal in need thereof an effective amount of a compound of claim
 1. 42.A method of treating or ameliorating hereditary tyrosinemia type 1 in ananimal, comprising administering to the animal in need thereof aneffective amount of a compound of claim
 1. 43. A method of treating orameliorating chronic alcohol ingestion induced buccal mucosa cell deathin an animal, comprising administering to the animal in need thereof aneffective amount of a compound of claim
 1. 44. A method of treating orameliorating cell death in plants or flowers, comprising administeringto the plants or flowers in need thereof an effective amount of acompound of claim
 1. 45. A method of treating or ameliorating radiationor ultraviolet-irradiation induced cell death in an animal, comprisingadministering to the animal in need thereof an effective amount of acompound of claim
 1. 46. A method of treating or ameliorating apoptoticdeath of bone marrow cells in myelodysplastic syndromes (MDS),comprising administering to the animal in need thereof an effectiveamount of a compound of claim
 1. 47. A method of treating orameliorating apoptotic cell death in acute pancreatitis, comprisingadministering to the animal in need thereof an effective amount of acompound of claim
 1. 48. A method of treating or preventing theinflammatory response in psoriasis or inflammatory bowel disease,comprising administering to the animal in need thereof an effectiveamount of a compound of claim
 1. 49. A method of treating orameliorating organ apoptosis after burn injury, comprising administeringto the animal in need thereof an effective amount of a compound ofclaim
 1. 50. A method of treating or ameliorating small bowel tissueinjury after intestinal ischemia-reperfusion, comprising administeringto the animal in need thereof an effective amount of a compound ofclaim
 1. 51. A method of treating, ameliorating or preventing oralmucositis, gastrointestinal mucositis, bladder mucositis, proctitis,bone marrow cell death, skin cell death or hair loss resulting fromchemotherapy or radiation therapy of cancer in an animal, comprisingadministering to the animal in need thereof an effective amount of acompound of claim
 1. 52. The method of claim 51, wherein said compoundis administered topically or orally.
 53. The method of claim 52, whereinsaid compound is formulated as part of a mouthwash for the treatment,amelioration or prevention of oral mucositis.
 54. The method of claim52, wherein said compound is formulated as part of a slow release buccallozenge.
 55. The method of claim 52, wherein said compound is formulatedas part of a suppository.
 56. The method of claim 52, wherein saidcompound is formulated as part of a gel.
 57. The method of claim 52,wherein said compound is administered through a bladder catheter for thetreatment, amelioration or prevention of bladder mucositis.
 58. Themethod of claim 52, wherein said compound is administered as part of anenema for the treatment, amelioration or prevention of proctitis. 59.The method of claim 52, wherein said compound is formulated as an oralformulation which is capable of coating the gastrointestinal surfacesfor the treatment, amelioration or prevention of gastrointestinalmucositis.
 60. The method of claim 59, wherein said gastrointestinalmucositis is esophageal mucositis, gastric mucositis, or intestinalmucositis.
 61. The method of claim 51, wherein said compound isadministered by i.v. injection for the treatment, amelioration orprevention of bone marrow cell death.
 62. The method of claim 51,wherein said compound is administered as part of a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier.
 63. Themethod of claim 51, wherein said compound is administered afterchemotherapy or radiation therapy of cancer in said animal.
 64. Themethod of claim 51, wherein said compound is administered duringchemotherapy or radiation therapy of cancer in said animal.
 65. Themethod of claim 51, wherein said compound is administered prior tochemotherapy or radiation therapy of cancer in said animal.